Avian influenza virus belongs to the orthomixoviridae family, and causes damage to poultry such as chicken, turkey. Avian influenza viruses are classified into 3 types of high-pathogenic, low-pathogenic and non-pathogenic avian influenza viruses according to the degree of pathogenicity, among which the high-pathogenic virus is classified as an OIE List A disease by the World Organization for Animal Health (OIE) and “a category 1 domestic animal infectious disease” in Republic of Korea.
Influenza virus is classified as type A, B or C according to the antigenicity of nucleocapsid protein and matrix protein. Moreover, according to the difference of antigen structure of haemagglutinin (HA) and neuraminidase (NA), the HA is classified into 16 subtypes and NA is classified into 9 subtypes, wherein HA helps host cell receptor binding, and fusion between host cell membrane and viral envelope to cause virus infection and NA plays an important role when virus buds out through the cell membrane after proliferation. Theoretically, 144 kinds of virus subtypes could exist by the combination of two proteins.
Infection generally occurs by contact with contaminated secretions, furthermore, and it could be spread through the air, in both particle and droplet forms, human feet, feed delivery vehicles, apparatuses and feces on the surface of eggs etc. Although there are various symptoms according to the pathogenicity of infecting virus, generally, they are respiratory symptoms, diarrhea and a sharp decline in egg production etc. Moreover, in some cases, cyanosis appears in the head region such as crest, edema appears on the face, or feathers are ruffled. Mortality rate also varies from 0% to 100% according to pathogenicity, but since the symptoms are similar to those of Newcastle Disease, infectious larynogotracheitis, mycoplasma infection and the like, an accurate diagnosis is required.
High pathogenic avian influenza had occurred 23 times from 1959 to 2003 throughout the world, most of them were endemic and contained. The outbreaks of highly pathogenic avian influenza subtype H5N1 had occurred in Korea in December 2003, occurred in more than 30 countries including Europe, Africa and most countries in Southeast Asia such as Japan, China, Thailand, Vietnam and Indonesia and thus have become pandemic. Although it is known that humans cannot become infected with avian influenza, prevention of avian influenza is of paramount importance to public health sector since the case of human infection with H5N1 in 1997, isolation of H9N2 avian influenza viruses from humans in 1999 in Hong Kong and human cases of H7 avian influenza infection in 2004 in Canada. According to a report of the World Health Organization (WHO), (http://www.who.int/csr/disease/avian_influenza/country/cases_table—2006—06—20/e n/index.html), it was confirmed that 228 persons had been infected with H5N1 subtype viruses and 130 persons of them died during the period of 2003 to Jun. 20, 2006 in 10 countries. In Korea, since an outbreak of low pathogenic avian influenza by H9N2 subtype viruses had occurred in 1996 and it reoccurred in 1999.
If an avian influenza outbreak occurs, in most countries, the poultry needs to be disposed of, and countries where avian influenza outbreaks have occurred cannot export poultry products, thus causing swingeing damages to poultry industry.
Furthermore, when there is a risk of human infection, the damages spread to the whole industry including the tourism industry and the transport industry, thus causing astronomical loss.
Natural substance refers to substances which are minimally processed without artificial ingredients, and the natural substances classified as GRAS (Generally Recognized As Safe) can be used without restrictions on the quantity thereof or foods in which the natural substances are to be used. In domestic industry, the natural substances are classified as natural additives, and used as food additives, and in foreign countries, it has been used as health foods and medical supplies for user's purpose without extra limitation, because of its excellent functionality.
Meanwhile, Alnus japonica is a deciduous, dicotyledonous tree in the order Fagales, family Betulaceae, which is commonly called Alnus japonica tree. They are distributed in Korea, Japan, China, etc., and grow in marsh conditions, its height is about 20 m and its bark is a deep purplish-brown color. Its winter bud is a long oval shape just like the shape of an egg turned upside down, which has three lines and a peduncle. The leaves of Alnus japonica grow alternately, and they are oval shaped, egg-shaped (more or less round on both ends, widest at the bottom) or lanceolate. Both sides of a leaf are lustrous and leaf margins are saw-toothed. The flowers of Alnus japonica bloom in March˜April, are unisexual, and form a catkin. Staminate spike bears staminate flower and each bract subtends 3˜4 flowers. There are four perianths and four stamens in each flower. Fruit ripens in October and 2˜6 fruits are produced. It is long egg-shaped and looks like a pine cone.
Examples of conventional patents relating to Alnus japonica extracts include a cosmetic composition containing an Alnus japonica extract (Korean Patent Publication No. 10-2003-0074500) and a method for preparing a health drink useful for relieving hangovers, which comprises extracts of Alnus japonica and green tea leaves (Korean Patent Publication No. 10-2006-0023093), etc.
Recently, many research endeavors are taking place to develop anti-viral agents throughout the world. Lamibudine used for the treatment of HIV (Human Immunodeficiency Virus)-1 and hepatitis B, gancyclovir used for the treatment of symptoms of herpes virus infection, ribavirin which is used mainly for the treatment of symptoms of respiratory syncytial virus infection but can be used for the treatment of symptoms of various virus infection when it is an emergency and zanamivir RELENZA™ and oseltamivir TAMIFLU™ which are synthesized artificially as influenza virus neuraminidase inhibitors are all commercially available after gaining approval. However, use of amantadine and its analogue, rimantadine, which are approved for treatment of influenza virus A, has decreased due to the appearance of resistant virus and its side effect. Recently, virus resistant to oseltamivir among H5N1 avian influenza viruses appeared, therefore, there is an urgent need to develop various antiviral agents.
The present inventors have confirmed antiviral activity of methanol extract of Alnus japonica in Korean Patent Registration No. 10-0721703 and Korean Patent Registration No. 10-0769050. However, the above mentioned patents have a disadvantage of showing antiviral activity only when the extracts were administered at high concentration and thus the possible applications thereof are limited.
Therefore, the present inventors have made an extensive effort to develop a natural substance having a low toxicity to normal cells, while having an excellent effect of inhibiting influenza virus proliferation even when administered at low concentration, and as a result, confirmed that an extract obtained by extracting the bark or stem of Alnus japonica, which is indigenous to Korea, with 8˜90% ethanol at 30˜80° C. , has an excellent anti-influenza virus effect, thereby completing the present invention.